Figure 4. Slit regulates filopodium... | Rockefeller University Press

Figure 4.

$Figure 4. Slit regulates filopodium dynamics by inducing a direct interaction between Mena EVH1 and Robo CC2. (A) Western blots of anti-Robo1 immunoprecipitates (IP) from CAD cells after stimulation with 5 nM Slit at the indicated times. Robo1 was not detectable in the unenriched input fraction. (B) Domain diagrams of Robo1/2 and Robo1-derived constructs used for pull-down experiments. (C) GST-EVH1 was used to pull down GFP-tagged fragments of the Robo intracellular domain from HEK293 cell lysates; robust interaction was detected with GFP-CC2 (asterisk). (D) GST-EVH1 pull-down from lysates of HEK293 cells expressing GFP-CC2 or GFP-CC2 in which the EVH1 binding site was mutated (CC2m, L>A substitution). Pull-down and unbound fractions displayed are from the same Western blot exposure; irrelevant lanes have been removed for clarity. (E) Densitometric analysis of GST-EVH1 pull-down of Robo intracellular domain (ICD) fragments containing the CC2 motif (mean ± SD, three independent experiments). **, P < 0.001; ***, P < 0.0001.$

Slit regulates filopodium dynamics by inducing a direct interaction between Mena EVH1 and Robo CC2. (A) Western blots of anti-Robo1 immunoprecipitates (IP) from CAD cells after stimulation with 5 nM Slit at the indicated times. Robo1 was not detectable in the unenriched input fraction. (B) Domain diagrams of Robo1/2 and Robo1-derived constructs used for pull-down experiments. (C) GST-EVH1 was used to pull down GFP-tagged fragments of the Robo intracellular domain from HEK293 cell lysates; robust interaction was detected with GFP-CC2 (asterisk). (D) GST-EVH1 pull-down from lysates of HEK293 cells expressing GFP-CC2 or GFP-CC2 in which the EVH1 binding site was mutated (CC2m, L>A substitution). Pull-down and unbound fractions displayed are from the same Western blot exposure; irrelevant lanes have been removed for clarity. (E) Densitometric analysis of GST-EVH1 pull-down of Robo intracellular domain (ICD) fragments containing the CC2 motif (mean ± SD, three independent experiments). **, P < 0.001; ***, P < 0.0001.